Effects of iron supplementation on iron status, health and neurological development in marginally low birth weight infants

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Umeå University Medical Dissertations, New Series No 1481

Effects of iron supplementation on

iron status, health and neurological

development in marginally low birth

weight infants.

Staffan Berglund

Department of Clinical Sciences, Pediatrics Umeå University

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Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) Correspondence: staffan.berglund@pediatri.umu.se

ISBN: 978-91-7459-371-6 ISSN: 0346-6612 New series No: 1481

Cover illustration: “Essential elements” by Nils Berglund. E-version available at http://umu.diva-portal.org/ Printed by: Print & Media

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Abstract

Background Due to small iron stores and rapid growth during the first months of life, infants with low birth weight (LBW) are at risk of iron deficiency (ID). ID in infancy is associated with irreversible impaired neurodevelopment. Preventive iron supplementation may reduce the risk of ID and benefit neurodevelopment, but there is also a possible risk of adverse effects. More than 50% of all LBW infants are born with marginally LBW (MLBW, 2000-2500g), and it is not known if they benefit from iron supplementation.

Methods We randomized 285 healthy, Swedish, MLBW infants to receive 3 different doses of oral iron supplements; 0 (Placebo), 1, and 2 mg/kg/day from six weeks to six months of age. Iron status, during and after the intervention was assessed and so was the prevalence of ID and ID anemia (IDA), growth, morbidity and the interplay with iron and the erythropoetic hormones hepcidin and erythropoietin (EPO). As a proxy for conduction speed in the developing brain, auditory brainstem response (ABR) was analyzed at six months. In a follow up at 3.5 years of age, the children were assessed with a cognitive test (WPPSI-III) and a validated parental checklist of behavioral problems (CBCL), and compared to a matched reference group of 95 children born with normal birth weight.

Results At six months of age, the prevalence of ID and IDA was significantly higher in the placebo group compared to the iron supplemented infants. 36% had ID in the placebo group, compared to 8% and 4 % in the 1 and 2mg/kg/day-groups, respectively. The prevalence of IDA was 10%, 3% and 0%, respectively. ABR-latencies did not correlate with the iron intake and was not increased in infants with ID or IDA. ABR wave V latencies were similar in all three groups. Hepcidin correlated to ferritin and increased in supplemented infants while EPO, which was negatively correlated to iron status indicators, decreased. At follow up there were no differences in cognitive scores between the groups but the prevalence of behavioral problems was significantly higher in the placebo group compared to those supplemented and to controls. The relative risk increase of CBCL-scores above a validated cutoff was 4.5 (1.4 – 14.2) in the placebo-group compared to supplemented children. There was no detected difference in growth or morbidity at any age.

Conclusion MLBW infants are at risk of ID in infancy and behavioral problems at 3 years of age. Iron supplementation at a dose of 1-2 mg/kg/day from six weeks to six months of age reduces the risks with no adverse effects, suggesting both short and long term benefit. MLBW infants should be included in general iron supplementation programs during their first six months of life.

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Original papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals (I-IV)

I. Berglund S, Westrup B, Domellöf M. Iron supplements reduce the risk of iron deficiency anemia in marginally low birth weight infants. Pediatrics. 2010;126(4):e874-e83.

II. Berglund SK, Westrup B, Haraldsson E, Engström B, Domellöf M. Effects of iron supplementation on auditory brainstem response in marginally LBW infants. Pediatr. Res. 2011; 70(6): 601-606.

III. Berglund S, Westrup B, Lönnerdal B, Domellöf M. Effects of iron supplementation on serum hepcidin and serum erythropoietin in low birth weight infants. Am J Clin Nutr. 2011;94(6): 1553-1561.

IV. Berglund SK, Westrup B, Hägglöf B, Hernell O, Domellöf M. Effects of iron supplementation of LBW infants on cognition and behavioral problems at 3 years of age - a randomized controlled trial. Umeå 2011. Submitted.

Paper I is reprinted with permission from Pediatrics Copyright © 2010 by the AAP, paper II with permission from Pediatric Research Copyright © 2011 by Wolters Kluwer Health and paper III with permission from American journal of clinical nutrition Copyright © 2010 by the American society for nutrition.

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Abbreviations

ADHD Attention deficit/hyperactive disorder AGA Appropriate for gestational age

ANOVA Analysis of variance CBCL Child behavioral checklist

EBF6w Exclusively breastfed at 6 weeks of age EPO Erythropoietin

Hb Hemoglobin

ID Iron deficiency

IDA Iron deficiency anemia LBW Low birth weight (< 2500 g)

MCV Mean corpuscular volume

MLBW Marginally low birth weight (2000-2500g)

OR Odds ratio

RR Relative risk

SD Standard deviation

SGA Small for gestational age

TfR Transferrin receptor concentration

TS Transferrin saturation

VLBW Very low birth weight (< 1500g)

WPPSI Wechsler preschool and primary scale of intelligence Z-score Standard deviation score

Keywords

Auditory brainstem response, behavior, breast feeding, cognition, erythropoietin, ferritin, growth, hemoglobin, hepcidin, human infant, iron, iron deficiency, iron deficiency anemia, iron status, iron supplementation, low birth weight, morbidity, neurodevelopment, nutritional requirements, randomized controlled trial.

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Populärvetenskaplig sammanfattning

Bakgrund

Järn är en livsnödvändigt mineral för kroppen. Det behövs både i kroppens röda blodkroppar, ansvariga för syretransport, men också i många proteiner i kroppens alla celler. Globalt sett är järnbrist ett mycket vanligt bristtillstånd framför allt hos små barn. Anledningen är att små barn växer proportionellt fortare och då behövs också mer järn.

Långvarig järnbrist ger blodbrist, eller lågt blodvärde (Hb) vilket ganska enkelt kan mätas genom blodprov. Men innan blodbrist uppstår kan det under lång tid finnas brist på järn, utan att det syns på blodvärdet. Detta stadium av järnbrist är svårare att upptäcka, eftersom våra olika indikatorer i blodet är svåra att tolka. Nyligen upptäcktes ett nytt protein, hepcidin, som skulle kunna vara en bra markör för att bedöma tillgången på järn i kroppen. En anledning till att järnbrist har blivit en stor fråga över hela världen är att flera tidigare studier har visat ett samband mellan tidig järnbrist och senare sämre neurologisk utveckling. Man tror att det är bristen på järn i hjärnan som kan vara förklaringen och dessvärre har några av dessa hjärnskador visat sig vara långvariga. Det är därför viktigt att förebygga järnbrist. Men situationen är mer komplicerad då flera ganska nya studier har visat att om man försöker förebygga järnbrist hos de som aldrig hade någon ökad risk att få det, ja då kan man få negativa effekter, till och med försämrad neurologisk utveckling.

En grupp barn med särskild risk för järnbrist är de som föds med låg födelsevikt. De barnen rekommenderas järntillskott för att inte utveckla tidig järnbrist. Den största gruppen av barn med låg födelsevikt har bara marginellt låg födelsevikt (2000-2500g). Vad gäller denna grupp är det oklart om de behöver järntillskott, eller om järntillskott bara ställer till problem.

Metod

Vi har genomfört en randomiserad studie där vi lottat 285 barn med marginellt låg födelsevikt till tre doser av järntillskott, givet under första levnadshalvåret. En grupp fick inget järn (Placebo) och två grupper fick järn i olika dos (1 respektive 2 mg/kg kroppsvikt och dygn). Vi har sedan undersökt barnen vid olika åldrar, senast vid 3.5 års ålder, för att se vilka

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effekter järntillskott hade på blodprover, tillväxt och den neurologiska utvecklingen, mm.

Resultat

I det första arbetet visade vi att barnen som inte fick något järn hade ökad risk för järnbrist (36%) och järnbrist med blodbrist (10%) vid sex månaders ålder. Bland dem som fick 2 mg/kg/dygn hade bara 4% järnbrist och ingen hade järnbrist med blodbrist. Risken var extra stor för de som fick sin huvudsakliga näring via bröstmjölk, eftersom bröstmjölk innehåller lite järn. I det andra arbetet undersökte vi om man kunde uppmäta skillnader i hörselnervens ledningshastighet (hjärnstamsaudiometri). Andra har föreslagit att den kan påverkas av järnbrist. Vi fann inga sådana skillnader utan konstaterade att hörselnervens ledningshastighet inte påverkades. I det tredje arbetet utvärderade vi de olika mätmetoderna för järnbrist inklusive den nya variabeln hepcidin. Hepcidin har föreslagits vara ett viktigt protein i regleringen av järn i kroppen och vi kunde visa att hepcidin påverkas av järnbrist och järnöverskott, även hos nyfödda. Även det blodbildningsstimulerande hormonet EPO undersöktes och vi fann ett intressant samband med järntillgång i kroppen.

I det fjärde delarbetet utvärderade vi barnen vid 3.5 års ålder avseende IQ och beteendeproblem för att se om den tidiga järnbristen påverkat hjärnan. Vi fann inga skillnader i IQ mellan de olika grupperna men däremot en skillnad i förekomst av beteendeproblem. Barn som inte fått järn under första levnadshalvåret hade 4 gånger ökad risk att ha beteendeproblem, då de skattades av föräldrarna med hjälp av en ofta använd enkät.

Slutsatser

Från denna studie drar vi slutsatsen att barn med marginellt låg födelsevikt har ökad risk att få järnbrist under första levnadshalvåret och att järntillskott kan skydda mot det utan att medföra några negativa effekter eller biverkningar. Detta skyddar inte bara mot järnbrist mätt med blodprover utan minskar också risken för beteendeproblem i förskoleåldern. Vår studie är den första randomiserade studie som visat ett samband mellan tidig järnbrist och senare beteendeproblem.

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Background

IRON METABOLISM

Iron is the fourth most common element in the earth crust and thought to be the main content of the earth core. Iron is also fundamental for life. Every adult human carries about 3.5-4 g of iron, representing about half a cubic centimeter. Yet this small amount of mineral, relative to the body weight, is essential, being a key building block in several proteins, distributed to three main compartments of the body:

Body compartments of iron

Oxygen transport

The main compartment of iron in the adult human (~30 mg Fe/kg body weight) is in the circulating blood, where each mature erythrocyte carries about 300 million hemoglobin molecules, containing four heme proteins, with one Fe2+ atom each. The oxygen carrying heme proteins are also represented by in myoglobin, the protein responsible for oxygen storage and transport in muscles (~4 mg Fe/kg).

Other tissue proteins

A smaller but yet as important fraction of the body iron (~2 mg Fe/kg) is present in various tissues as iron containing enzymes and other proteins. The complete list of functional iron-proteins is not understood, but iron is essential in electron transport proteins present in all body cells (i.e. cytochromes), activators of molecular oxygen (i.e. pexoxidases and catalases), and many others.

Storage

The third compartment of iron is the storage iron, making the body prepared for unexpected loss or changes in iron absorption. This is a “buffer” compartment, were large changes can occur between a state of iron depletion (~1-2 mg/kg) and repletion (~5-30 mg/kg), without affecting the functional iron compartments. The main storage proteins are ferritin and hemosiderin. Ferritin is found essentially in the liver, spleen, bone marrow, and muscles and each molecule can bind 2500-3000 iron atoms. In a well designed interplay between ferritin-containing cells and the transport protein

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transferrin, a small amount of iron is portioned to circulating plasma, bound to transferrin addressed to the various tasks of iron in the body.1-4

The flux of iron

Iron absorption

Mammals have no mechanism for excreting iron. However, small passive losses (~14µg/kg/day) occur daily from cells lost in the gastrointestinal- and urinary tracts, as well as from the skin. Adding the losses from occasional small bleedings and for women menstrual bleedings, an average loss of 1-2 mg; representing 15-40 µg/kg/day is present in a basal state. Thus, this is also the required mean daily absorption that must take place to maintain iron homeostasis. Furthermore, if sudden losses occur, such as large bleedings from wounds, occult bleedings from tumors, gastric ulcers, and gastrointestinal infections, the absorption must be further up-regulated. Iron is available in the intestine as heme-bound iron and non-heme iron, depending on type of diet. For heme-iron, representing about 10% of dietary intake, the mechanisms of absorption are poorly understood. However the absorption of heme-iron is 6-7 times more effective compared to non-heme iron, making it an important source. For non-heme iron the absorptive mechanisms are better understood: Non-heme iron is reduced to Fe2+ by ferrireductases in the brush border of the enterocyte. When reduced, the iron molecule is transported into the enterocytes via the divalent metal transporter 1 (DMT1). Much of the iron absorbed into the enterocytes is stored there, bound to ferritin and only a fraction is transported through the basolateral membrane by the transport protein ferroportin. This part of the transport is subject to regulation. Since enterocytes are at constant renewal and old cells are lost into the gastrointestinal lumen, ferritin bound iron, not transported further to the plasma will be lost with the desquamated cells.1-4

Erythrocyte-bound iron

Once transported to the lumen by ferroportin, iron binds to transferrin, which has two binding sites for iron. About 350µg/kg/day of transferrin-bound iron is utilized in the bone marrow and used for erythropoiesis. This represents a daily ten times turnover of the transferrin bound iron, which at any moment is only about 35µg/kg. The transferrin-iron complex has high affinity to transferrin receptor 1 (TfR), located on the surface of the developing erythroid cell. The complex of TfR, transferrin and iron is absorbed into the cell by a well described, essential cycle, ending in incorporation of the iron molecule into the heme protein and secretion of the

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empty transferrin protein back to the blood stream. The regulation of erythropoiesis is mainly mediated by erythropoietin (EPO), a hormone produced in the kidney as a response to tissue hypoxia.

Erythrocytes have a life cycle of ~120 days and old cells are phagocytosed by macrophages, mainly in the spleen (MPS-cells). The MPS-cells store iron bound to ferritin and release it to the blood by ferroportin, the same transport protein as enterocytes. The macrophage release of iron is the main source of circulating transferrin bound iron (~300µg/kg/day).1-4

Other targets of transferrin-bound iron

The transferrin-bound iron not used for erythropoiesis is transported in similar manner to other cells in need of iron. If the circulating iron exceeds the needs, it is incorporated into storage cells, mainly in the liver hepatocytes. Also hepatocytes use TfR receptors as capturers of iron and ferroportin for excretion. For tissues separated from the blood such as the brain, the transport mechanisms are not yet understood. Transferrin bound iron cannot cross the blood-brain barrier and thus, other mechanisms must be involved. The proportions of iron compartments and the utilization of iron are summarized in Figure 1.

Muscles (Myoglobin) Blood (Hemoglobin) Other tissues (proteins etc.) Liver, spleen, macrophages (Ferritin) Losses (14‐40µg/kg) Plasma (35µg /kg ) 350 µg Intestinal lumen Absorption (14‐40µg/kg) 350 µg 350 µg DMT1 ? Heme‐Fe Fe3+ _Fe2+ Fe 3+ _ferroportin Tissues (2mg/kg) Oxygen transport (30‐35mg/kg) Storage (2‐30mg/kg)

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Iron homeostasis

The total body iron is regulated by changing iron absorption in the intestine and it has been shown, in adults, that the DMT1 and the ferroportin-transporters are correlated strongly to iron status.5_{Furthermore, iron}

absorption is regulated by the degree of erythropoiesis and the rapid utilization between iron compartments indicates a well designed regulation. Yet, the actors of iron homeostasis and particularly their signaling and regulation are not fully understood. However, during the last decade, rapid progress has occurred within this research field, not least due to the discovery of the iron regulatory protein hepcidin.6-7

Hepcidin

Hepcidin is produced in the liver, released into the blood stream and acts as an inhibitor of ferroportin (figure 2).8_{Through this mechanism it can down}

regulate the release of iron from the enterocytes in the intestinal tract, the iron storage cells in the liver, muscles and spleen, or particularly the large flow of iron from the MPS-cells, trapping iron from phagocytosed erythrocytes.

Regulation of hepcidin

Most research on hepcidin is based on cellular or animal models and the clinical experience is poor. As of today there are five separately described, sometimes opposing regulators of hepcidin.1-4

1. Transferrin saturation. Hepcidin synthesis increases as response to increased transferrin-bound iron, thus maintaining a relatively constant level of circulating iron. The mechanism by which this occurs is not fully understood, but includes the transferrin receptor 2 (TfR2), the HFE protein and the co-receptor HJV, all three associated with the disease

hemochromatosis, characterized by an inappropriate iron absorption and tissue incorporation.

2. Iron stores. In a yet even less well understood pathway, including the protein BMP6, iron stores in the hepatocytes are integrated with the signal of transferrin saturation. Thus, a dual input both from iron stores and circulation iron affects hepcidin expression.

3. Erythropoietic signals. Hepcidin is reduced as a response to increased erythropoiesis, suggesting that the largest consumer of iron can affect absorption and cellular release. The association between iron absorption and erythropoiesis was known before the discovery of hepcidin. This association is of particular clinical interest since it has been suggested that

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the hepcidin inhibiting effect from erythropoiesis is more powerful than the stimulating effect from iron stores, and might be an explanation why patients with hemolytic diseases, who typically has increased erythropoiesis and replete iron stores (iron loading anemias, e.g. thalassemia), suffer from poor down regulation of iron absorption.9

4. Inflammation. Hepcidin is increased in inflammation. The mechanisms have been partly described and include the cytokines Interleukin 1 and 6, which interact with hepatocytes and stimulate hepcidin expression. The physiological effect is that circulation iron is reduced to starve a potential microbe from iron. Clinically, this sometimes causes a problem in cases of prolonged autoimmunity and malignancy, where the reduced iron availability causes anemia of inflammation.

5. Hypoxia. In cellular models, hepcidin has also found to be decreased in situations of hypoxia, assumed to be in concordance with the effect of erythropoiesis.

Liver

Plasma

Spleen

Bone

marrow

Intestinal

lumen

Fe‐Tf Fe‐Tf Fe‐Tf Fe‐Tf Fe‐Tf Fpn Fpn Fpn Hep Hep Hep

‐

Fe‐Tf Fe‐Tf Hep Hep Hep Hep

‐

Erythropoiesis

+

Iron stores Inflammation Iron sensing

Figure 2. Illustration of the iron regulatory effect of hepcidin (Hep) and its regulation. Hepcidin expression in liver cells is stimulated by circulation transferrin bound iron (Fe-Tf), iron stores and inflammation and down regulated by erythropoetic activity and ID. Hepcidin down-regulates ferroportin (Fpn) in enterocytes, and iron- storing cells.

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Diagnostic tools of iron status

From a clinical view point the ability to evaluate iron status in an individual is of great importance. There are several diseases associated with disorders of iron metabolism, and there is a need of reliable diagnostic tools. Depending on the question, several of the actors in iron metabolism could contribute with such information.10-12

Ferritin

Functionally being the protein involved in iron storage, ferritin is a common tool for assessing iron stores, and the most accepted.12_{Even though the}

underlying mechanism is not understood it is a fact that the level of serum ferritin is in equilibrium with body stores and thus used as an indirect measure. It is estimated that one µg/L ferritin corresponds to 120µg storage Fe/kg body weight.11_{Ferritin concentration declines early in a state of iron}

deficiency (ID) and serum levels below 12-15µg/L indicates depleted stores and increased levels indicate iron overload.10_{A limitation in the use of}

ferritin is that it is also an acute phase reactant, increasing in states of inflammation, unrelated to iron status. Another limitation is that it does not indicate the degree of functional iron depletion.10-12

Iron, transferrin and transferrin saturation

Transferrin is increased in ID and serum iron levels are reduced. However there is a diurnal variation and the variables should be interpreted with caution, particularly since transferrin-bound iron represents less than one tenths of a percent of iron in the body. Calculating transferrin saturation (TS), i.e. the ratio of serum iron and the total binding capacity of transferrin (TIBC), gives a better but yet not completely reliable measure of available serum iron. TS is used both as an indicator of ID and iron overload.10-12

Serum transferrin receptors (TfR)

Erythropoetic cells and other cells in need of iron signal by increased TfR concentration at the cell surface. Similar to ferritin, a proportional amount is assumed to be circulating in serum and can be measured. Increased TfR is associated with early tissue iron need. Further, it has been suggested that TfR concentration is not affected by inflammation, making it an important complement to ferritin. However international standards are missing and the assays are expensive. Furthermore, reference values in infants and children are scanty.10-12

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Mean corpuscular volume (MCV)

As iron availability in erythropoiesis reaches a critically low level, erythrocyte production is maintained but with decreased hemoglobin content, resulting in decreased erythrocyte cell size, measured as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), or mean corpuscular hemoglobin concentration (MCHC), all late signs of ID.10-12

Hemoglobin (Hb) and hematocrit

Since most of the body iron is bound to hemoglobin, a state of prolonged ID will result in decreased hemoglobin levels and erythrocyte volume fraction (EVF or hematocrit). Traditionally, hemoglobin measures have become the key measure of ID and international standards, reference- and cutoffs-levels are available, though they are repeatedly questioned.12_{However, being an}

indirect measure if ID, anemia (low hemoglobin) should be interpreted with cautions and other causes considered.10-12

Other indicators

Erythrocyte protoporphyrin (EPP) is the precursor of heme and becomes

elevated when iron availability is low.10-13

Erythrocyte zinc protoporphyrin (ZPP) is produced as zinc replaces missing

iron in the formation of heme. It has a high sensitivity but poor specificity to identify ID.10-12

Bone marrow iron staining has been considered golden standard of

measuring iron stores. However the procedure is far too complicated to be used clinically. 10-12

Hemoglobin response to iron supplementation is a common practice to

evaluate if present anemia is associated to ID. An increase of 10g/L in hemoglobin indicates prior deficiency. But the method has been questioned, particularly in infants.10-12, 14

TfR/ferritin ratio. The logarithm of this ratio has been suggested to correlate

better than any individual measure to total body iron.15_{However its advantage} over ferritin alone has been questioned.10-12

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Iron deficiency (ID)

ID is considered present when available iron supply for physiological function is inadequate. It is the most common micronutrient deficiency in the world and a public health problem.10_{The World Health Organization}

(WHO) estimates that the global prevalence of anemia is 25% (47% in preschool aged children) and advocate that ID is the most significant contributor.16-17_{However estimating the prevalence of ID in the world or}

even in subgroups of populations is difficult, since the measures of functional supply are indirect, and difficult to interpret.12, 15

The process of ID-development theoretically progress through three stages: 1. Storage iron depletion

2. Early functional deficiency (or iron deficient erythropoiesis) 3. Established functional deficiency (or iron deficiency anemia)

Separating two stages of functional deficiency underlines that there could be impaired tissue availability, before the Hb level declines, causing a risk of damage in other functional compartments of iron, i.e. the brain.12_The

challenge lies in identifying this stage. Storage iron depletion Early functional deficiency Established functional deficiency Ferritin ↓ ↓↓ ↓↓ TS Normal ↓ ↓↓ TfR Normal ↑ ↑↑ MCV Normal (↓) ↓

Hemoglobin Normal Normal (↓) ↓

EPP Normal ↑ ↑↑

ZPP Normal ↑ ↑↑

Hb response to suppl. None (↑) ↑↑

Table 1. Iron status indicators and their suggested change during stages of ID.

Biomarkers of iron deficiency

There is an ongoing debate among researchers on which biomarkers best identify ID, particularly the second stage. In screening programs anemia alone is commonly used, although most agree that it is not specific, since anemia may result from other causes. Neither is it sensitive since ID in the two earlier stages by definition is overreached.12_{Since functional deficiencies}

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are of most clinical concern, also ferritin has been questioned, being a marker of iron stores and not tissue availability. Furthermore, as mentioned, in presence of inflammation, ferritin cannot be trusted.10-12, 15_{For remaining}

indicators, there are several pros and cons but a common problem is lack of international standards and established cut offs. Instead, the use of combinations of 3-4 biomarkers has been suggested, defining ID when 2 out of 3 or 2 out of 4 indicators are abnormal.12_{Table 1 summarizes the available}

indicators of iron status and their theoretical change during stages of ID.

Iron metabolism in infants

Much of what is known of iron metabolism is based on research of adults and cannot be applied directly on children and particularly not on infants. The reasons for this are several.

In opposite to the stable distribution between iron compartments in adults, the rapidly growing infant experiences both large redistributions and an overall increase of tissue bound iron. Iron requirements are not restricted to the iron needed to compensate for small losses from skin and gastrointestinal tract, but also include the iron needed in the rapid expansion (growth) of erythroid mass and other tissues. Assuming a mean growth rate of 24 g/day during the first six months of life18_{and 35 mg of functional iron}

per kg body weight corresponds to an iron requirement of ~150 µg/kg per day, compared to the estimated 15-40 µg/kg/day assumed in adults (see above).

To meet this, the infant with normal birth weight is born with elevated iron stores. The average iron content of a newborn is about 75 mg/kg,19

decreasing to about 45 mg/kg at one year of age.20_{By redistribution of the}

iron compartments, these iron stores are enough to meet the needs during the first 4-6 months of life, with no exogenous iron needed, explaining why breastfed infants do not develop ID during their first half year of life; even though human milk has low iron content.5

During this redistribution, not only are iron stores decreasing compared to the newborn. Also the hemoglobin concentration is decreasing during the first 6-8 weeks of life, making further iron available to the expanding erythropoetic mass and other growing tissues.21-22_{Nevertheless, iron stores}

will be close to depleted at 4-6 months of age and requirements of exogenous iron rapidly increase during the second half year of life, making term infants, at particular risk of ID from about six months of age (see below).20, 23

Figure 3 summarizes the redistribution of iron assumed during the first 12 months of life.

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0 50 100 150 200 250 300 350 400 450

Birth 6 wk 6 mo 12 mo

mg

Tissues

Oxygen transport Storage

Figure 3. Total body iron and its proportional distribution between the main compartments during the first year of life. Reproduced from Domellöf et al.24_with

permission from S. Karger AG.

Infant iron regulation

Our understanding of the homeostatic mechanisms in infant iron metabolism is poor. Mostly knowledge are based on animal models and studies on adults.23_{The validity of this approach has not been confirmed.}

Since there is a rapid, physiological change in iron compartments and erythropoiesis, the infant’s homeostasis is clearly different from that of adults and regulatory mechanism could theoretically be different, or even absent. For instance, it has been suggested that the ability to up regulate iron absorption, develop between six and 9 months of age and that it prior to six months of age is immature.5, 25_{Such immature regulation might be}

physiological since it would allow iron stores and erythropoetic mass to rapidly change without affecting absorption and cellular release, but it would also put infants at particular risk of iron overload.5_{The role of hepcidin in}

infant iron metabolism has not been evaluated.14_{Theoretically, absence or}

poor sensitivity for hepcidin could explain the immaturity suggested.26

Assessing infant iron status

Defining what is “normal” iron status in the newborn infant is challenging due to rapid physiological changes.23_{The levels of hemoglobin and}

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reference ranges must take age into account. Most commonly, iron status reference ranges have been constructed by examining the range in healthy infants, some exclusively breastfed and others given iron fortified formula or supplements, making the results diverging. However, for infants aged less than six months, such descriptive trials are few and the suggested ranges wide. The correlation with functional outcomes is not known.14, 23_Still,

hemoglobin and ferritin are considered most efficient at least in monitoring effect of iron supplementation, but their cutoff values have been questioned.5, 27

IRON DEFICIENCY IN INFANTS

Due to the rapid growth rate and high requirements, ID is the most common single nutrient deficiency in infants.10, 16_{Since ID without anemia is difficult}

to assess (see above), the exact prevalence is unknown. In developed countries, with well nourished children, the prevalence of IDA is < 3-4% 28-30

However the prevalence of ID among US children aged 12-35 months has been estimated to 9%, and several risk groups are identified, with even higher prevalences.31

Infants and children at increased risk of ID

Exclusively breastfed infants

As discussed above, exclusively breastfed infants are at risk of having depleted their iron stores at about 4-6 months of age.23_{If iron-enriched}

complementary food, infant formula or follow on formula is not added to the diet at that age; there is a substantial risk of established functional ID. At what age exogenous iron, either as iron fortified complementary food or supplementation, should be introduced into the infant’s diet is under debate.23, 32-33_{Several studies have suggested that this risk is not present}

before six months of age, in healthy normal birth weight infants, and WHO does recommend exclusive breast feeding until six months of age, based on the numerous benefits that is associated with breastfeeding.28, 30_However,

recently the American Academy of Pediatrics suggested that breastfed infants are at risk already from four months of age and recommended supplemental iron of 1 mg/kg/day to all breastfed infants receiving more than one half of their daily feeding as human milk, a recommendation that has been questioned.31, 33

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Preterm and LBW infants

Iron stores accumulate rapidly in the fetus during the third trimester to reach about 75mg/kg at term birth.19, 34_{Since this process in not complete}

when born before term, preterm infants are born with lower iron stores proportional to the body weight.35_{Additionally, the postnatal proportional}

growth rate is mostly more rapid in preterm compared to term infants and preterm infants thus empty their stores of iron more rapidly.10_Similarly,

SGA infants are considered at increased risk due to the rapid catch up growth. There are also trials suggesting lower iron stores in SGA, term infants.36-37_{Recommendations for preterm/LBW infants are reviewed below.}

Prenatal ID

Several factors are associated with fetal/neonatal ID.

Severe maternal ID is the most common cause of prenatal ID.38-39_{Cord blood} ferritin as a proxy of iron stores are correlated to the degree of maternal iron status. Infants of anemic mothers, even if not ID at birth, are at increased risk of postnatal ID.5, 39

Maternal diabetes increases fetal iron demand due to rapid fetal growth and

increased erythropoiesis due to hypoxia.38-40

Maternal smoking and maternal hypertension are other known risk factors.35

Possible disadvantages of infant ID

As mentioned, anemia is the most common marker of ID. The symptoms of anemia are mostly none, but fatigue, palpitations, headache, dizziness, pale skin and insomnia are typical symptoms of severe anemia in adults. If present in infants, they constitute possible disadvantages. Anemia is also associated with impaired growth and lower work capacity in children and adults. But anemia is not the only manifestation of ID. Also immune dysfunction, pica, breath holding spells and restless leg syndrome are associated with ID.17_{However of most concern is that ID in infants and}

children is associated with impaired neurodevelopment.

ID and brain development

Iron is essential for neurogenesis and in differentiation of brain cells. Simultaneously, brain growth is rapid during the last trimester of pregnancy and during the first two years of life, even more rapid than the rest of the

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body. Not surprisingly, research in animal models, mostly rodents, has shown an association between ID in infancy and impaired brain development and function.38-39, 41-43

The effects on brain development are depending on timing, duration and severity of ID.38, 41-42_{Several of the alterations in the brain, observed in}

animal models of ID are long lasting and do not reverse if iron therapy is added.39_{The existence of a critical window of irreversible damage has been}

suggested.43_{Furthermore, it has been suggested that iron is prioritized for}

erythropoiesis over other functions, and consequently, functional deficits in the brain may appear before IDA occurs.17, 38-39_{There are three domains of}

brain function, that are most studied in ID animal models.

Neurotransmitter metabolism. ID alter the synthesis and catabolism of

neurotransmitters such as dopamine, serotonin and norephenephrine.39, 42 Dopamine is important in regulation of higher functions such as cognition, emotion, reward and motivated behavior particularly active in striatum and the basal ganglia.41-42_{In rodent models of moderate ID, dopamine and} serotonin metabolism are altered, proportionally to the degree of ID. Some of these alterations persists into adulthood 39, 41_{There is also evidence of} behavioral alterations in these rats such as delayed sensory-motor reflexes, less exploration and more hesitancy, all associated with dopamine functions.39, 41_{Recent studies in monkeys with infant ID showed similar} effects of prolonged lower dopamine levels.39

Morphological alterations and dendritic structure. ID causes decreased

dendritic growth and reduces the interneuronal connections in hippocampus, a structure central to recognition and memory.38-39, 42_{The findings are} accompanied by behavioral alterations (learning and trace conditioning), and persists into adulthood despite iron therapy.38, 41

Myelination. Oligodendrocytes, the cells that produce myelin, are particularly

sensitive to ID. In rodent models, ID causes altered composition and amount of myelin in large regions of the brain and these changes persists.39, 42

Human studies

When applying the knowledge of ID and brain development from animal models on human infants, the situation becomes more complicated. Behavioral development is multi-factorial. An association between ID and development might be explained both by a direct damage to brain tissues and by an indirect effect on the child’s exploratory behavior or parental interactions.44_{An association can also be confounded by disadvantages in}

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family structures, causing both increased risk of ID and behavioral impairment, without a direct causality. Figure 4 summarizes the possible associations between ID and child behavior as summarized by Osendarp.43

Below follow a summary of available research in human infants and toddlers with or at risk of ID.

Disadvantaged Environment Level of child involvement with the environment Iron deficiency Brain development and function Quality of Parent‐child interactions Behavior development

Figure 4. Possible explaining causalities of the association between iron deficiency and child behavior. Reproduced from Osendarp43_{with permission.}

Evidence of impaired neurological development

There are a substantial number of trials and reports on the effect of IDA in infants and children. Fewer have examined infants with ID without anemia, mostly due to difficulties in identifying them. Most are cross sectional or longitudinal case-controlled trials and few are randomized interventions of iron supplementation. Furthermore, most studies investigated the neurological functions at time of, or short after ID/IDA and compared to controls but few have performed follow up studies, to assess long term consequences. There are several reviews of published trials.44-46

Case-controlled trials

An association between IDA and neurological performance has been suggested in several of these trials. Most commonly, studies have shown impaired mental (cognitive) or motor achievements in infants and young children with IDA compared to controls.39, 45-46_{In trials evaluating cognitive}

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scores, a difference of 6-15 points was seen in infants with IDA compared to iron replete infants.45_{Differences in behavioral and social-emotional}

development has also been reported. IDA infants are found less pleased, more hesitant, less playful and with functional isolation.44-45_{In trials}

performed on infants with prenatal ID (infants of diabetic mothers), infant were found to have poorer recognition memory and behavioral tests.45_From

these case-controlled trials, a few include follow-up after treatment of the IDA. Most but not all reported persisting deficits in some domains of neurodevelopment, but the follow up period was mostly short.44-45

More important from a public health perspective are the assessments of long-lasting effect by early ID and IDA. In follow ups of pre-school and school aged children with IDA in infancy, a few case-controlled trials suggest evidence of long-lasting impairment in cognition, behavior, motor development and increased educational problems.45_{In a trial on Costa Rican}

infants, impaired cognitive and behavioral achievement was found at follow-up at five, 11-14 and 19 years of age in those who were diagnosed with IDA in infancy, compared to a iron replete control group.47-49

A limitation of these case-control trials is that that infants with IDA are more likely to have socio-demographic risk factors and these can confound the results (figure 4). Although researchers have tried to statistically control for these confounders, uncertainty still remain of causality between ID and poor development, and the association has been questioned.44-45

Neurophysiologic outcomes

To improve research of the association between ID/IDA and neurodevelopment, a few trials have analyzed neurophysiologic functions. Auditory brainstem response (ABR) is a measure of conduction speed in the auditory nerve, and a proxy of myelination of neurons. Case controlled-trials of infants with IDA compared to controls have shown prolonged ABR-latencies, suggesting an impaired myelination of the neurons.50-51_{In one}

trial, the impaired latencies persisted, and even magnified at follow-ups until four years of age, suggesting an irreversible damage.51-52_Other

case-controlled trials have shown alterations in EEG, eye-blinking patterns, and visual evoked potentials, all attributed to IDA.45

Randomized controlled trials

Some randomized placebo-controlled trials (RCT) have analyzed the effect of iron supplementation of infants with already existing IDA. A few of those have shown a positive effect on mental, motor or language development. But

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again, the results are diverging, possibly depending on the time, duration and severity of ID/IDA or the time and duration of supplementation.44_{In a}

recent meta-analysis, Sachdev et al concluded that there is evidence of a small but significant benefit on mental development, but not in motor development.53_{However they included all ages and found that the effect was}

lesser in young children and infants. As for mild/moderate ID without anemia there is insufficient data.44

RCTs of preventive iron supplementation are few. In a recent meta-analysis, only five randomized trials assessing neurological development following preventive iron supplementation to infants below 3 years of age was identified.54_{The authors concluded that none showed benefits in mental}

development while three trials showed positive effect on psychomotor development.55-59_{None presented follow up data above 18 months of age.}

Similarly, Grantham-McGregor recently concluded that there is evidence of improved psychomotor development but not enough evidence to state causality between iron supplementation and improved cognitive development in infants and small children. With respect to long term effects, there is no available data.44

Benefits and risks of iron supplementation

Given the suggested association between ID and impaired neurological development, iron supplementation is recommended to infants diagnosed with, or at risk of ID.10, 60_{However humans have no ability to excrete iron}

and iron, being a pro-oxidant, is a potentially toxic drug. Thus, possible negative effects or overdosing must be considered, especially if supplementation or fortification is recommended in populations or parts of populations, where individual follow up is impossible.61_{Notably, such}

negative (adverse) effects have recently been reported, causing concerns in the field.61-62

Anemia

Most trials of iron supplementation to infants at risk of or with diagnosed IDA show increased hemoglobin levels. As discussed above, this has been suggested as a reliable measure of present IDA. However, an increase in hemoglobin concentration is in itself of no benefit to a child, unless it results in short or long term functional benefits.

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Growth

A few studies on ID infants have shown a positive effect of iron supplementation on growth63-64_{but a recent meta-analysis of 27 iron}

supplementation trials to children below five years of age showed no overall effect on growth.65_{What is of more concern is that four trials have suggested}

a negative effect on growth when analyzing iron replete infants separately, underscoring the importance of identifying the correct risk groups for supplementation.66-69 Less anemia Improved growth Improved motor development Cognitive development ? Behavior ? Impaired growth Increased infections Gastrointestinal problems ? Cognitive development ?

Benefits

In iron deficient infants

Risks

In iron replete infants

Figure 5. The balance of possible benefits and risks of iron supplementation to infants.

Infections

Iron is also an essential nutrient to pathogens i.e. bacteria, fungi and viruses. As part of the immune response in humans, circulating iron is reduced, to reach less iron available to pathogens. Theoretically, this mechanism can be broken during iron supplementation and it has been suggested that iron supplementation can increase the risk of infections, supported by results from some clinical trials.62_{Most recent evidence relate to the risk of Malaria.}

A large iron supplementation trial In Zanzibar had to be terminated due to increased risk of severe Malaria infection in supplemented infants.70_{Also in}

this study, the adverse effect was most pronounced in those initially iron replete.62

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Neurological development

The possible positive neurological effects of iron supplementation to infants at risk of ID were reviewed above. However a very recent trial, have suggested that iron supplementation might also have adverse effects on neurodevelopment. Healthy Chilean infants were randomized to infant formula with different levels of iron fortification from six to 12 months of age (12.7 mg/L vs. 2.3 mg/L). At 10 years, infants fed the high iron formula had significantly lower scores on visual motor integration and memory, and a non-significant trend of lower IQ and arithmetic achievement. The negative effects were not present in those with lowest initial hemoglobin levels, further supporting the hypothesis that iron replete infants are at risk of adverse effects when given excess iron.71

Summary

Iron supplementation can benefit millions of infants and possible improve neurodevelopment if given to those at risk of ID. But due to possible adverse effects, identifying the correct risk groups for general iron supplementation requires large randomized trials and correct interpretations of the outcomes. The balance of pros and cons is summarized in figure 5.

LOW BIRTH WEIGHT INFANTS

Low birth weight (LBW) is defined as birth weight less than 2 500g. Unicef estimated in 2009 the global prevalence to 14% and 7% in industrialized countries.72_{The prevalence in Sweden is 4.7% (Socialstyrelsen,}

unpublished).

A substantial amount of research and medical resources in industrialized countries, focus on the more severe forms of LBW, i.e. infants born very LBW (VLBW, < 1 500g) or extremely LBW (ELBW < 1 000g). As illustrated in figure 6, a majority of LBW infants are only moderately LBW (1500-2500g). Actually > 50 % of LBW infants have a birth weight of 2000-2500g, a group of infants rarely studies. Below we refer this group to marginally

low birth weight (MLBW). The situation is similar when studying age

categories of preterm infants. Of 12.3% preterm infants in US 2008, 8.8% were born late preterm (34-36 weeks of gestation).73

Marginally low birth weight infants (MLBW)

MLBW infants are a heterogeneous group, including infants born moderately preterm (31-33 weeks), late preterm (34-36 weeks) and term (> 37 weeks),

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each category to varying degree including SGA infants. The MLBW SGA infants can further be categorized into intrauterine growth restricted (IUGR), where the small size is caused by poor nutrition in late gestation, but also constitutionally small infants with genetic predisposition for small size.

Due to this heterogeneity, the knowledge of MLBW infants is poor since they are mostly excluded from clinical trials of both term and preterm infants. However during recent years, MLBW and late preterm infants have been more prioritized. One reason being that it has been suggested that also this group is at risk for increased mortality and morbidity, both perinatally and later in life.74-75 < 500 g 500‐999 g 1000‐1499 g 1500‐1999 g 2000‐2499 g 0% 1% 2% 3% 4% 5% 6% USA Sweden

Figure 6. Distribution of weight categories in US and Swedish LBW infants.

Morbidity in MLBW children

LBW is associated with increased disadvantages in life such as death during infancy and childhood, congenital anomalies, respiratory infections, neurodevelopmental impairment, school failure and growth problems.76

Even though the risk is lower in MLBW infants compared to those VLBW, it is still increased compared to normal birth weight infants.77

Neonatal morbidity

Late preterm and MLBW infants compared to full-term infants are at increased risk of feeding disabilities, temperature instability, hypoglycemia,

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respiratory distress, jaundice and septicemia.78_{The mortality rate is 3-fold}

higher than for term infants.74, 79

Neurodevelopmental impairment

SGA and preterm LBW infants are associated with cognitive disabilities, ADHD and other behavioral problems.80-84_{It has been suggested that LBW}

contributes to 14% of all cases of attention-deficit/hyperactivity disorder (ADHD).85_{The risks increase with decreasing gestational age and birth}

weight,82-84, 86_{However several recent trials have suggested that compared to}

term normal birth weight infants, also moderately LBW infants have increased risk if impaired neurodevelopment.80, 82, 86-89_{The increased risks}

have been attributed to perinatal complications such as intra-ventricular hemorrhage, sepsis, metabolic complications, painful procedures, and maternal separation. Another common explanation is that there are confounding socio-demographic factors causing both LBW and neurological problems.84, 90_{However the association between LBW and}

neurodevelopment persists in several trials even when adjusting for such confounders. 80, 82-83, 85-90

Metabolic and cardiovascular risk

Barker and colleagues showed in early 1990s that LBW was associated to cardiovascular disease and diabetes. The “Barker hypothesis” suggests that early fetal or perinatal starvation causes a disturbance in metabolic programming, resulting in increased morbidity later in life. Several trials have confirmed an association between LBW and obesity, cardiovascular disease, diabetes, hypertension, and other markers of metabolic syndrome and LBW is now considered a risk factor of later cardiovascular disease. 91-95

However a recent meta-analysis concluded that there is no overall risk of obesity in LBW children suggesting that the association only relates to subgroups.96_{Others have suggested that the growth acceleration, following}

prenatal or perinatal starvation might be the reason to inadequate metabolic imprinting, suggesting that the risk could be reduced by feeding interventions.97

Nutritional disadvantages

Except for the practical feeding problems present due to immaturity or during neonatal morbidity, LBW infants require special feeding interventions in several domains. Multi-component fortifier is recommended to infants born < 32 weeks of gestation and special pre-term formula to non-breastfed VLBW infants. However for MLBW and late preterm infants, WHO

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concluded in a report from 2006 that there is insufficient data to make recommendations on preterm infant formula content, Vitamin D, Phosphorus, calcium, Vitamin A, Zinc or multi-component fortifiers. For iron, WHO recommends supplementation from 6-8 weeks of age but conclude that there is a paucity of data on its effect on mortality, morbidity, and neurodevelopment particularly in MLBW infants.98

Iron requirements in LBW infants

As discussed above, preterm and SGA LBW infants theoretically deplete their iron stores earlier than term, normal birth weight infants due to rapid growth and lower iron stores at birth. This is supported by the clinical experience all around the world, where VLBW infants receive iron supplements to adjust iron stores and hemoglobin levels. Early research on bone marrow aspirates in preterm infants suggested that age of depletion is around 1-3 months of age followed by a fall in Hb-levels, referred to as late anemia (figure 7).34, 99_{However, evidence for beneficial effect of iron}

supplementation to LBW infants is limited to a few trials mostly from the 1960s - 1980s. The knowledge of dose, age to start supplementation, duration, as well as the long term consequences is unsatisfactory.98, 100_This

is of particular concern since it has been suggested that preterm infants exert no control over iron absorption and are at particular risk of iron overload.34

0 4 8 12 16 20 0 20 40 60 80 100 120 Hb konc (g/dL) Hb mass (g) Early anemia Late anemia Age (days) Iron stores

Figure 7. Development of anemia of prematurity has been suggested to appear i two steps, first the physiological drop in Hb-levels at birth and second a late phase of anemia suggested to be caused by depleted iron stores. Modified from Shaw et al.34

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Identified trials assessing iron supplementation to LBW infants are summarized in table 2. In brief, there is evidence from developed countries that LBW infants < 2000g are at increased risk of developing IDA during the first six months of life, and that iron supplementation starting at about 4-8 weeks of life, has a preventive effect.

MLBW and late preterm

MLBW infants are included in several trials but we found only one trial analyzing MLBW infants separately. In that, the prevalence of ID in MLBW breastfed infants was 75% at six months compared to 22 % in the group of infants fed iron-fortified formula (8mg/L). However, only 26 MLBW infants were analyzed.101_{In opposite, Halliday et al analyzed moderately and late}

preterm infants of 28-36 weeks of gestation and mean birth weight 2020g. They found no effects of iron supplementation from 7 days of age, or 8 weeks of age compared to placebo solution including only ascorbic acid.102_In

secondary analyses they compared late preterm infants (33-36 wk) with moderately preterm infants (28-32 wk), and found iron status changes during the first half of infancy to be very similar, suggesting that iron stores deplete similarly in the two subgroups.103_{In contrast, Lundström showed}

that risk of anemia increase with decreasing birth weight.104

Diverging recommendations

The paucity of conclusive data from MLBW infants is underscored by the diverging recommendations to this group. A recent questionnaire sent to all Swedish neonatal centers revealed that MLBW infants are prescribed iron in doses from 0 – 2.2 mg/kg/day during the first six months of life. None of the centers recommended prolonged supplementation beyond six months of age (unpublished). Similar disparity of policies were reported from a British survey of 57 neonatal units, 105_{and in a retrospective case-control trial of}

LBW infants in New Zeeland.106_{In 2003, the British recommendations were}

to restrict iron supplementation to LBW infants below 1800g,107_{while the}

American Academy of Pediatrics (AAP) recommend 2 mg/kg/day from 1-12 months of age to all LBW and preterm infants who are breastfed and 1 mg/kg/day to those formula fed.108_{The ESPGHAN Committee on nutrition}

recommended an intake of 2-3 mg/kg/day to preterm infants with no separate recommendations to late preterm or MLBW infants and without specifying how this could be interpreted in clinical interventions.109

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Study Included infants

Objective Outcome Result

James 1960110 205 LBW <2000g Intramuscular iron (250mg x 5) when body weight was 2000g vs. no treatment (Randomized) Hb from 1‐6 mo and incidence anemia  6 mo Sign diff. in Hb from 2‐6 mo** Incidence of anemia until 6 mo: 0 vs. 90%** Hammond 1960111 66 preterm 1300‐2500g Intramuscular iron (200‐ 400mg) vs. no treatment (Randomized) Iron status and incidence of Anemia  12 mo Sign diff. in Hb from 3‐12 mo* Incidence of anemia until 6 mo: 9 vs. 23%* Gorten 1964112 145 preterm mean 1.9kg Fortified formula (12mg/L) vs. non fortified formula from birth (Randomized) Hb from 1‐18 mo and incidence of Anemia  18 mo Sign. difference in Hb from 3*, 4 – 18 mo** Incidence of anemia until 6 mo: 2 vs. 35% * (Large drop‐out) Lundström 1977104 177 LBW breastfed, 1050‐2000g 2mg/kg/d oral suppl. from 2 wk to 6 mo vs. no iron. (Randomized) Hb, iron status and incidence of Anemia 6 mo Sig.n difference in Hb, MCV, TS, ferritin from 3 mo. Anemia in 0 % vs. 77% ** Halliday 1983102 49 preterm mean 2020g, 33.1 weeks 12mg/day from 7 days of age vs. 8 weeks vs. ascorbic acid (Randomized) Iron status  12 mo No sign. effect on Hb, iron, TS, transferrin or ferritin Iway 1986101 45 LBW 1000‐2499g (26 MLBW) 15 breastfed vs. 30 formula fed (8mg/L). (Case‐control) Iron status at 6 mo and incidence of ID 6 mo Sign. difference of Hb**, MCV*, Ferritin** and ID (86 vs. 33%)**. No difference in RBC, TIBC and TS. MLBW: 75% vs. 22% ID* Olivares 1992113 84 LBW mean 2194g 3 mg/kg/day oral from 2‐4 mo vs. no suppl. Stratified in 3 groups; Preterm/SGA, P/AGA, or Term/SGA (Randomized) Hb and ferritin at 4 mo Sign. difference in Hb and ferritin for Preterm/SGA and Preterm/ AGA*, but no difference in Term/SGA Hall 1993114 79 LBW formula fed, < 1800g Formula of 15 vs. 3 mg/L from 21 days until discharge. (Randomized) Iron status at discharge and 8 weeks post discharge Ferritin, MCV sign. higher at discharge* and 8 wks post** Prevalence low ferritin 8 weeks post discharge lower** Borigato 1998115 45 preterm mean 1935g Potatoes cooked in iron pots vs. aluminum ports from 4‐12 mo of age, added to suppl. of 2mg/kg/day (Randomized) Iron status and prevalence anemia at 12 mo Sign. effect on Hb*, hematocrit*, MCV*, EPP* and ferritin** Anemia in 36 vs. 74%* Griffin 1999116 81 preterm <1750g Infant formula of 5 vs. 9 mg/L (Randomized) Iron status at 6 mo No sign. differences Friel 2001117 58 LBW formula fed, < 2500g Formula of 20.7 vs. 13.4 mg/L from discharge (Randomized) Iron status and developmental index at 12 mo No sign. differences Thom 2003106 81 LBW < 2500g Low vs. high iron intake past month (Case control) Prevalence ID at 10 mo Sign. increased risk of ID if low iron intake. OR 13.4 ** Aggarwal 2005118 73 SGA, term Breastfed, <2500g 3mg/kg/day vs. no supplementation (Randomized) Hemoglobin at 4 wk, 8 wk, and after 2 mo Sign. difference at 4 wk and 8 wk* but not on follow up Franz 2000119 Steinmacher 2007120 133 LBW < 1301 g 2‐4 mg/kg/d as soon as enteral feeding tolerated vs. start at 61 d of age (Randomized) Ferritin and incidence ID at d 61. Neurodevelopment at 5.3y No difference in ferritin and 15 vs. 40% ID** at day 61. Non‐sign. trend of improved cognitive and psychomotor development at 5.3y. No effect on behavior.

Table 2. Identified randomized or case-controlled trials assessing iron supplementation to LBW and preterm infants. * p< 0.05. ** p< 0.001

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SUMMARY AND UNSOLVED QUESTIONS

Iron is essential to humans, distributed between three main compartments of the body; oxygen transport proteins, other tissue proteins and storage. The hormone hepcidin regulates the release from stores and enterocytes. Present diagnostic tools of iron status are difficult to interpret since they are collected from plasma or serum, which is only a proxy of what happens in the tissues in need of iron. This is unsatisfactory, since ID is the most common micronutrient deficiency, in need of trustable diagnostic tools.

Novel diagnostic tools of iron availability, particularly to detect early functional ID needs improvement and those present needs to be evaluated based on functional outcomes.

The iron homeostasis in infants is different from adults in needs of exogenous iron and in the proportions of compartments.

The mechanisms of iron regulation, the role of hepcidin and the validity of iron status indicators in infants are poorly known.

The need of iron for tissue growth in infants is ~10 times greater per kilogram than for adults and infants are at increased risk of ID. ID in infancy is associated with poor neurological development, supported primarily by research on animal models, but also from case control trials, showing an association of ID in infancy and impaired neurodevelopment. But since a disadvantaged environment might be a confounder of this association there is no undisputable causality proven. A few randomized trials have suggested causality between ID and impaired short term psychomotor development but none on cognition or behavior. Other trials have shown possible adverse effects on growth, infections, and even cognitive development if iron is given to those already replete.

There is no proven causality between ID in infancy and impaired long term neurodevelopment, particularly cognitive or behavioral.

LBW infants are the largest risk group of ID in developed countries. But this is confirmed only in those with moderately or very LBW (<2000g). The most common LBW infants are those born with marginally low birth weight (MLBW).

It is not known if iron supplementation benefit or harm MLBW infants.

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Objectives

The general objective of this trial was to test the hypothesis that marginally low birth weight infants (MLBW, 2000-2500g) benefit from iron

supplementation given during their first six months of life.

The specific objectives were to assess possible benefits and adverse effects of iron supplementation from six weeks to six months of age, given to healthy, Swedish infants, with regard to the following outcomes (respectively paper in parenthesis):

 Iron status, growth and morbidity until six months of age (I).  Auditory brainstem response at six months of age (II).

 Hepcidin, erythropoietin and its interplay with iron status from six weeks to six months of age (III).

 Cognitive and behavioral effects at 3.5 years of age (IV).

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Subjects and methods

PARTICIPANTS AND DESIGN

This was a prospective, randomized, placebo-controlled, double blinded trial including 285 healthy MLBW infants and 95 term, normal birth weight controls born in Umeå (n=44+15) and Stockholm (n=241+80) between March 2004 and November 2007. In brief, the 285 MLBW infants were stratified for sex and study center and randomized to three different doses of iron supplements, given as oral drops between six weeks and six months of age (Figure 8).

Age

6 wk 12 wk 19 wk 6 mo 12 mo 3.5 y

285 MLBW infants Placebo 1 mg/kg/day 2 mg/kg/day Data collection

Figure 8. Study design.

Study compliance

Of the included infants, 24 (8%) dropped out during intervention and another 18 (6%) until the follow up at 3.5 years. Compliance to the intervention was measured by a daily checklist, filled in by the parents and by weighing the bottles. In 62 cases (22%), the compliance to the intervention was <70%, of which 43 infants belonged to the iron supplemented groups. Poor compliance was equally distributed among the groups.

Discontinued cases

Infant iron status was assessed at six weeks and 12 weeks of age. Due to ethical considerations, infants diagnosed with anemia by then, were further evaluated by a pediatrician. Those with suspected IDA were prescribed iron supplementation and thereby discontinued the intervention. However, they continued as unblinded participants and were included in some analyses (see

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below). Thirteen infants were prescribed iron at six weeks and 9 infants at 12 weeks of age. Placebo (n=95) Analyzed at 3.5 y: (n=77) Analyzed at 3.5 y: (n=70) Analyzed at 3.5 y: (n=77) Analyzed at 6 mo: (n=78) Analyzed at 6 mo: (n=82) 2 mg/kg/d (n=95) 1 mg/kg/d (n=95) Analyzed at 6 mo: (n=83) Randomly assigned: (n=285) Dropouts: (n=9) Dropouts: (n=8) Excluded: Blood disorder: (n=1) Hb < 90 g/L: (n=2) Excluded: Blood disorder: (n=1) Hb < 90 g/L: (n=8) Dropouts: (n=7) Autism: (n=1) Dropouts: (n=5) Dropouts: (n=6) Dropouts: (n=8) Excluded: Hb < 90 g/L: (n=6)

Figure 9. Trial profile of the participants when analyzing according to the intention to treat principle in non anemic MLBW infants (Paper I, II and IV).

Exclusions and inclusions

The primary outcomes, presented in paper I, II and IV, assessed the group effect in infants non anemic at baseline (preventive trial). Thus the 16 infants with anemia at six weeks were excluded. The analyses were preformed according to the intention to treat principle, including also discontinued cases. During the intervention, another two infants were diagnosed with blood disorders (beta-thalassemia and ABO-immunization) and were excluded from all analyses. Another child was diagnosed with an autism-disorder (22q11-deletion syndrome) preceding the follow up and was excluded in analyses at 3.5 years. The trial profile in figure 9, illustrates the participant flow in these main analyses. In paper III and in some secondary analyses of the ‘per protocol’ group-effect, other inclusion and exclusion criteria were used as summarized in table 3.

Effects of iron supplementation on iron status, health and neurological development in marginally low birth weight infants